Pathogenic spirochetes most often associated with human Lyme disease have been grouped into three genospecies, B. burgdorferi, B. afzelii, and B. garinii. Interestingly, all three genospecies seem to populate different niches during persistent infection, which ultimately results in different clinical manifestations. For example, B. burgdorferi and B. afzelii are commonly associated with disease involving the joints and skin, respectively, while B. garinii typically causes central nervous system abnormalities. The underlying mechanism that results in the different tissue tropisms and disease manifestations between genospecies is not clear. However, it was recently shown that both B. burgdorferi and B. afzelii bind host serum complement inhibitor factor H on their cell surface, which confers complement resistance. By contrast, B. garinii does not bind factor H and is complement sensitive, which has led to the hypothesis that B. garinii organisms persistently infect the central nervous system because it is an environment limited in cytotoxic complement. Recently, several B. burgdorferi outer surface lipoproteins (Osp) were identified by us and others that can bind factor H in vitro. The factor H binding proteins identified include several lipoproteins related to OspE as well as a borrelial protein designated BbA68. To further these prior studies, and more directly examine the actual role that factor H-binding proteins play in complement resistance, we inactivated the bbA68 gene in an avirulent strain of B. burgdorferi. While the wild type parental strain is fully complement resistant, the mutant that lacked expression of BbA68 was found to be highly complement sensitive, suggesting that this protein may be the major molecule that imparts complement resistance to B. burgdorferi. To further examine the contribution of BbA68 in complement resistance of Borrelia spp. during an actual infection (i.e., in vivo), we propose to (i) inactivate the bbA68 gene in a virulent strain of B. burgdorferi and (ii) over express BbA68 in a virulent, complement sensitive B. garinii isolate. The mutant strains generated will then be examined for changes in virulence and/or disease pathogenesis using the mouse model of Lyme disease. The combined studies outlined in this R21 application focus on providing the necessary foundation and preliminary data for a future R01 application that will examine the functional role of BbA68 in spirochete tissue tropism and Lyme disease pathogenesis. [unreadable] [unreadable]